Blood collection devices, including tubes, are, used to collect blood to produce serum or plasma which is in turn used for biochemical or other pathology assays.
Serum is produced by allowing the blood sample to clot and then centrifuging the sample to separate the blood clot including cells from the serum. Plastic tubes (in place of glass) are now typically used and require procoagulants (often micronised silica particles) to enhance the clotting process. Serum is usually preferred over plasma for biological testing unless urgent results are required, in which case the clotting time for a serum tube is considered too long. Even with existing procoagulants, in most commercial tubes the minimum required clotting time recommended by manufacturers is 30 minutes for blood samples from normal patients, and much longer (typically 60 minutes or longer) for samples from patients taking anti-clotting therapeutic agents such as warfarin or heparin. For patient samples from emergency situations (emergency departments, intensive care, operating theatres etc.) the time is too long and therefore plasma, which can be produced much faster, is often preferred over serum. An alternative purported to address this issue is a blood collection tube for serum production recently developed by Becton-Dickinson (designated BD Rapid Serum Tube, BDT or BD RST) which contains thrombin designed to increase the rate and extent of blood clotting in blood samples.
Plasma is formed by collecting blood in tubes containing anticoagulants followed by centrifugation which can be performed immediately after collection to separate the cells and thus obtain plasma for analysis. Lithium heparin is the most commonly used anticoagulant in these tubes. Citrate, sodium fluoride/potassium oxalate and EDTA are other anticoagulants that are used in some tubes to produce plasma for estimation of a small number of other analytes.
Incomplete Clotting
The coagulation process in preparing a serum sample consumes fibrinogen and entraps platelets and other cells within a network of fibrin. Upon centrifugation the serum is separated from the clot, either by serum separator in the collection device or by aliquoting the serum into a secondary container, to prevent contact with cells. This separation permits the sample to remain stable for extended periods of time. This stability is particularly important if samples are not analysed immediately, or if re-analysis or additional analyses are required.
For some serum samples, coagulation is incomplete after the recommended waiting times. This problem of incomplete clotting is especially prevalent in patients on anti-clotting therapy or specimens collected from anticoagulated taps or cannulae. Contamination of the specimen with anticoagulant agents during collection may also occur. Such blood can take much longer than the manufacturer's recommended waiting time to clot, or in fact may never fully clot in a standard serum tube (e.g. blood from cardiac surgery patients who are fully heparinised). If a serum sample is centrifuged before clotting is complete, clotting can continue in the serum, leading to clots, microclots or formation of fibrin strings capable of causing analyser or analyte specific problems. The formation of microclots and fibrinogen strings during sample preparation may also occur in plasma tubes, especially post-storage at low temperatures. Lack of timely inversion of lithium heparin tubes after blood collection can lead to small clot formation around the rubber stopper. Droplets of blood not heparinised in a timely manner will clot, and clots do not disintegrate upon heparinisation.
Even the smallest clots are capable of producing clinically significant errors. Thus for accuracy, samples must be manually checked by eye or using automated detection systems if available to ensure they are free of fibrin strands or clots. If strands or clots of insoluble material are present, the sample requires sub-sampling into a new container and re-centrifugation prior to test analysis. Samples that exhibit repeated latent clotting may need to be transferred to a lithium heparin tube to stop ongoing clotting. These actions take additional time. Further, fibrin strands or clots are not always detected (e.g. they may even occur post analyser sampling), and consequential sampling errors may lead to patient care decisions being made on inaccurate results.
Cell Contamination in Plasma Tubes
Specimens obtained in plasma tubes, lithium heparin plasma specifically, may be contaminated with cells. Lithium heparin gel tubes when centrifuged will always present a small “buffy coat like layer” on top of the gel at the bottom of the plasma. This layer contains fibrin, cells and cell stroma. The rapid gel movement during centrifugation leaves some cells in the plasma. If the plasma specimen is mixed (e.g. during sub-sampling or handling), it will become turbid due to suspension of cell-containing material and fibrin, which decreases the specimen integrity. In addition, platelet aggregates can form which may also contain fibrin and/or white blood cells. These aggregates can be large enough to be visible to the unaided eye and have been termed “white particulate matter” due to their typical white colour, and present similar problems to incomplete clotting discussed above.
The presence of cells in the sample can affect analyte concentrations. Certain analytes (e.g. glucose) may be decreased by cell activity and others may be increased by leakage or cell lysis (e.g. lactate dehydrogenase, potassium, phosphate).
Analyte Interference
Although generally there is no difference in concentration of analytes measured in serum or plasma tubes, there are some exceptions.
Plasma tubes that use heparin are not suitable for heparin analysis or cell-based assays. Lithium heparin plasma tubes are not suitable for lithium analysis. Plasma may be unreliable for additional testing of re-testing, due to presence of cells and insoluble fibrin formation upon prolonged storage at 2-8° C.
Further, there have been reports of some serum or plasma tubes producing inaccurate results of analyte levels, due to interaction with the procoagulant or anticoagulant agents within the tubes, or otherwise (Ciuti et al., 1989; Cowley et al., 1985; Davidson et al., 2006; Dimeski et al., 2004; Dimeski et al., 2005; Dimeski et al., 2010; Hartland et al., 1999; Miles et al., 2004; O'Keane et al., 2006; Wannaslip et al., 2006).
Sample Size
It is desirable to reduce the sample size needed for testing, especially in critically ill patients, patients receiving blood transfusions, and infants, in order to reduce the volume of blood taken from a patient. It is therefore optimal to be able to run all necessary tests using a sample taken in a single blood collection tube. To achieve this, testing methods have been developed using very small sample volumes (e.g. 2 μL) so that typically one serum or plasma tube is used for at least 21 tests, but can be used for between 50-60 or even 70-80 tests, depending on the volume of sample needed for each test. However, where there is doubt over the accuracy of measuring a particular analyte in a serum or plasma tube, it may be necessary to take both a serum tube and a plasma tube from the patient and doing so defeats the goal of reducing the volume of blood taken from the patient.
Problems arising from the use of current methodologies for serum and plasma preparation from blood show that improvements are required to achieve timely, reliable analytical results from a wider variety of blood samples generally.
Snake Venom Prothrombin Activators
Many snake venoms contain prothrombin activators for the purpose of rapid clotting of the blood of their prey. These prothrombin activators are proteolytic enzymes which convert prothrombin present in blood to thrombin which in turn causes clotting.
While snake venom prothrombin activators are known procoagulants, they are also known to possess proteolytic trypsin-like activity (Schieck et al., 1972; Parker, H. W. and Grandison A. G. C., 1977; Masci, P. P., 1986; Nicholson et al., 2006; Lavin and Masci, 2009). It has been postulated that there may be an evolutionary reason that prothrombin activators possess both procoagulant and proteolytic properties in that they act to both kill and digest the prey (Masci, P. P., 1986, page 143). For example, ecarin (prothrombin activator purified from Echis carinatus venom) has been shown to have procoagulant activity and as well several other proteolytic activities such as fibrinogenolysis, gelatinolysis, caesionlysis and haemorrhage (Schieck et al., 1972), and a prothrombin activator purified from the venom of Pseudonaja textilis (PtPA) is active against a range of chromogenic peptide substrates designed for different proteolytic enzymes (Masci, P.P., 1986).
Many analyte tests that may be performed on blood, serum, or plasma samples involve proteins, including tests measuring proteins as analytes (e.g. total protein, albumin); tests measuring enzyme activity of blood proteins (e.g. gamma-glutamyl transpeptidase used in test for gamma-glutamyl transferase, aspartate aminotransferase, lactate dehydrogenase, creatine kinase, lipase); tests using proteins as reagents (e.g. immunoassays); tests using enzymes in the analytical method (e.g. glucose oxidase). Other commonly used tests involving protein include assays for glucose, urea, urate, alanine aminotransferase, creatine kinase, high-density lipoprotein cholesterol, cholesterol, triglycerides, transferrin, C reactive protein, troponin, cortisol, free thyroxine, free triiodothyronine, thyroid stimulating hormone, and ferritin.
Therefore, despite their procoagulant properties, these snake venom prothrombin activators have never been considered suitable for use in serum tubes for analyte tests, on the basis that their proteolytic activity would degrade analytes being measured (e.g. where the analyte is a protein), or would degrade proteins being used in the reaction to measure analyte levels (e.g. where the analyte test involves use of a protein such as glucose oxidase).
Thrombin Tubes
While thrombin-containing tubes have recently become available as ‘faster’ clotting tubes, and thrombin possesses both procoagulant and proteolytic activity, thrombin is known to have high specificity for cutting bonds in fibrinogen, activated protein C (APC) and Factor Va. Therefore, unlike the reported trypsin-like activity of the snake venom prothrombin activators, thrombin would not be expected to interfere with analyte tests.
In work leading up to the present invention, it was found that thrombin-containing tubes cannot be used with all blood samples. Thrombin is known to be rapidly and completely inhibited by the heparin-antithrombin III complex present in heparinised blood samples. In investigating the BD RST tubes, it was found that these tubes are ineffective in clotting patient samples containing high doses of heparin (Dimeski et al., 2010).
Development of the Invention
Surprisingly, the present inventors discovered that when used in blood collection devices, including tubes, prothrombin activators are generally capable of producing high quality serum in an acceptable time from a wide variety of blood samples (including those taken from patients on high concentration of anti-clotting therapy, including heparin), decreasing both the serum sample preparation time and the risk of analysis problems due to incomplete clotting and contamination by cells and cell components.
Moreover, the inventors also surprisingly discovered that serum samples obtained from blood samples by addition of prothrombin activators give the same results in a wide range of standard biochemistry analytical tests as serum samples produced in existing blood collection tubes.
These discoveries suggested that prothrombin activators would be suitable for producing serum for the purpose of measuring a wide range of analytes, and have been reduced to practice in blood collection containers for preparing serum samples useful in detecting analytes, related uses and methods, as described hereafter.